Tissue samples were homogenized in 1X tissue lysis buffer containing a protease and phosphatase inhibitors cocktail (Roche cOmplete Tablets, Mini EDTA-free, # 04 693 159 001) and incubated on ice for 15 min. Lysates were cleared by centrifugation at 12,000 g and 4°C for 15 min. After determining protein concentration using BCA protein assay kit (Thermo Fisher/Pierce), 30 μg of protein lysates were mixed with Laemmli buffer, heated at 95°C for 5 min, and cooled on ice for 3 min. The samples were resolved on 4–15% Mini-PROTEAN TGX gels (Bio-Rad) under reducing conditions and blotted on to PVDF membranes using Trans turbo system (Bio-Rad). Membranes were blocked with odyssey TBS blocking buffer (Licor) and immunoblotted overnight at 4°C with a specific antibody against B1R (#ABR-011, Alomone labs). The membranes were then incubated with the corresponding secondary antibody, and bands were visualized using the Odyssey Clx imager (Licor). The density of protein bands were quantitatively analyzed by ImageJ software (version 1.52p, NIH) and expressed as a relative ratio against the loading control.
Transturbo system
Manufactured by Bio-Rad
The TransTurbo system is a high-performance laboratory equipment designed for various scientific applications. It functions as a centrifuge, providing efficient separation and purification of samples.
Automatically generated - may contain errors
Lab products found in correlation
Tgx gel, by Bio-Rad (4 mentions)
Substrat hrp immobilon western, by Merck Group (4 mentions)
Complete tablets mini, by Roche (1 mentions)
Odyssey blocking buffer tbs, by LI COR (1 mentions)
Odyssey clx imager, by LI COR (1 mentions)
Mini protean tgx gel, by Bio-Rad (1 mentions)
Image lab, by Bio-Rad (1 mentions)
5 protocols using transturbo system
1
B1R Receptor Protein Quantification
2
Protein Analysis by Immunoblotting
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Protein analysis by immunoblotting was performed essentially as previously described27 (link). Briefly, samples were collected, washed out with PBS and lysed with RIPA buffer. Protein concentration was determined by BCA assay (Pierce) before electrophoresis in 4–15% TGX gels (BioRad) and equal amount of protein was loaded in each well. Protein transfer was performed in TransTurbo system (BioRad) in PVDF membranes. After blocking for 1 h with 5% non-fat milk, membranes were incubated overnight at 4 °C in agitation with primary antibodies, washed three times with PBS-Tween 0,1% and incubated with the appropriate HRP-labeled secondary antibody for 1 h. Membranes were washed out three times with PBS-Tween 0,1% and developed with Substrat HRP Immobilon Western (Millipore). Band quantification was performed using the “ImageLab” software from BioRad and represented as the ratio between the protein of interest and a control protein i.e. actin. The value of 1 is arbitrarily given to control cells. One blot representative of several experiments is shown.
3
Immunoblotting for Protein Analysis
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Protein analysis by immunoblotting was performed essentially. Briefly, samples were collected, washed out with PBS and lysed with RIPA buffer. Protein concentration was determined by BCA assay (Pierce; Dallas, TX, USA) before electrophoresis in 4–15% TGX gels (BioRad) and equal amount of protein was loaded in each well. Protein transfer was performed in TransTurbo system (BioRad) in PVDF membranes. After blocking for 1 h with 5% non-fat milk, membranes were incubated overnight at 4 °C in agitation with primary antibodies, washed three times with PBS-Tween 0.1% and incubated with the appropriate HRP-labeled secondary antibody for 1 h. Membranes were washed out three times with PBS-Tween 0.1% and developed with Substrat HRP Immobilon Western (Millipore). Band quantification was performed using the “ImageLab” (version 3.0.1) software from Bio-Rad and represented as the ratio between the protein of interest and a control protein, i.e., actin. The value of 1 is arbitrarily given to control cells. One blot representative of several experiments is shown.
4
Immunoblotting Protein Analysis Protocol
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Protein analysis by immunoblotting was performed essentially as previously described (Garaude et al., 2006 (link), Garaude et al., 2008 (link)). Briefly, samples were collected, washed out with PBS and lysed with RIPA buffer. Protein concentration was determined by BCA assay (Pierce) before electrophoresis in 4–15% TGX gels (BioRad) and equal amount of protein was loaded in each well. Protein transfer was performed in TransTurbo system (BioRad) in PVDF membranes. After blocking for 1 h with 5% non-fat milk, membranes were incubated overnight at 4 °C in agitation with primary antibodies, washed three times with TBS-Tween 0,1% and incubated with the appropriate HRP-labeled secondary antibody for 1 h. Membranes were washed out three times with TBS-Tween 0,1% and developed with Substrat HRP Immobilon Western (Millipore). Band quantification was performed using the “ImageLab” software from BioRad and represented as the ratio between the protein of interest and a control protein i.e. actin. The value of 1 is arbitrarily given to control cells. One blot representative of several experiments is shown.
5
Immunoblotting for Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein analysis by immunoblotting was performed essentially as previously described45 (link). Briefly, samples were collected, washed out with PBS and lysed with RIPA buffer. Protein concentration was determined by BCA assay (Pierce) before electrophoresis in 4–15% TGX gels (BioRad) and equal amount of protein was loaded in each well. Protein transfer was performed in TransTurbo system (BioRad) in PVDF membranes. After blocking for 1 h with 5% non-fat milk, membranes were incubated overnight at 4 °C in agitation with primary antibodies, washed three times with PBS-Tween 0,1% and incubated with the appropriate HRP-labeled secondary antibody for 1 h. Membranes were washed out three times with PBS-Tween 0,1% and developed with Substrat HRP Immobilon Western (Millipore). Band quantification was performed using the “ImageLab” software from BioRad and represented as the ratio between the protein of interest and a control protein i.e. actin. The value of 1 is arbitrarily given to control cells. One blot representative of several experiments is shown.
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